The present invention relates generally to the biosynthesis of glycans found as free oligosaccharides or covalently bound to proteins and glycosphingolipids. This invention is more particularly related to a family of nucleic acids encoding UDP-D-galactose:xcex2N-acetylglucosamine xcex21,3-galactosyltransferases (xcex23Gal-transferases), which add galactose to the hydroxy group at carbon 3 of 2-acetamido-2-deoxy-D-glucose (GlcNAc). This invention is more particularly related to a gene encoding the fifth member of the family of xcex23Gal-transferases, termed xcex23Gal-T5, probes to the DNA encoding P3Gal-T5, DNA constructs comprising DNA encoding xcex23Gal-T5, recombinant plasmids and recombinant methods for producing xcex23Gal-T5, recombinant methods for stably transfecting cells for expression of xcex23Gal-T5, and methods for indication of DNA polymorphism in patients.
A family of UDP-galactose; xcex2-N-acety -glucosamine xcex21-3galactosyl-transferases (xcex23Gal-T""s) was recently identified (Amado, M., Almeida, R., Carneiro, F., et al. A family of human xcex23-gal actosyltransferases: characterisation of four members of a UDP-galactose xcex2-N-acetylglucosamine/xcex2-N-acetylgalactosamine xcex21,3-Galactosyltransferase family. J. Biol. Chem. 273:12770-12778, 1998; Kolbinger, F., Streiff, M. B. and Katopodis, A. G. Cloning of a human UDP-galactose:2-acetamido-2-deoxy-D-glucose xcex23-galactosyltransferase catalysing the formation of type 1 chains. J. Biol. Chem. 273:433-440, 1998; Hennett, T., Dinter, A., Kuhnert, P., Mattu, T. S., Rudd, P. M. and Berger, E. G. Genomic cloning and expression of three murine UDP-galactose: xcex2-N-acetylglucosamine xcex21,3-galactosyltransferase genes. J. Biol. Chem. 273:58-65, 1998; Miyaki, H., Fukumoto, S., Okada, M., Hasegawa, T. and Furukawa, K. Expression cloning of rat cDNA encoding UDP-galactose G(D2) xcex21,3 galactosyltransferase that determines the expression of G(D1b)/G(M 1)G(A1). J. Biol. Chem. 272:24794-24799, 1997). Three genes within this family, xcex23Gal-T1, -T2, and -T3, encode xcex23gal actosyltransferases that form the Galxcex21-3GlcNAc linkage. The type 1chain Galxcex21-3GlcNAc sequence is found in both N- and O-linked oligosaccharides of glycoproteins and in lactoseries glycosphingolipids, where it is the counterpart of type 2 Galxcex21-4GlcNAc poly-N-acetyllactosainine structures (Kobata. A. Structures and functions of the sugar chains of glycoproteins. Eur J Biochem 209:483-501, 1992.). Type 1 chain structures are found mainly in endodermally derived epithelia, whereas the type 2 chains are found in ecto- and mesodermally derived cells including erythrocytes (Oriol, R., Le Pendu, J. and Mollicone, R. Genetics of ABO, H, Lewis, X and related antigens. Vox Sanguinis 51:161-171, 1986; Clausen, H. and Hakomori, S. ABH and related histo-blood group antigens; immunochemical differences in carrier isotypes and their distribution. Vox Sanguinis 56:1-20, 1989). Normal gastro-intestinal epithelia express mainly type 1 chain glycoconjugates, while type 2 chain structures are predominantly expressed in tumors (Hakomori, S. Aberrant glycosylation in tumors and tumor-associated carbohydrate antigens. Tumor malignancy defined by aberrant glycosylation and sphingo(glyco)lipid metabolism. Advances in Cancer Research 52:257-331, 1989; Hakomori, S. Tumor malignancy defined by aberrant glycosylation and sphingo(glyco)lipid metabolism. Cancer Res 56:5309-5318, 1996). It is of considerable interest to define the gene(s) responsible for formation of these core structures in normal and malignant epithelia. Several characteristics of the three previously described xcex23Gal-Ts capable of forming type 1 chain structures suggest that these are not the major enzyme(s) involved in type 1 chains synthesis in epithelia: (i) Northern analysis indicates that xcex23Gal-T1 and -T2 are exclusively expressed in brain (Amado, M., Almeida, R., Carneiro, F., et al. A family of human xcex23-galactosyltransferases: characterisation of four members of a UDP-galactose xcex2-N-acetylglucosamine/xcex2-N-acetylgalactosamine xcex21,3-Galactosyltransferase family. J. Biol. Chem. 273:12770-12778, 1998; Kolbinger, F., Streiff, M. B. and Ktopodis, A. G. Cloning of a human UDP-galactose:2-acetamido-2-deoxy-D-glucose xcex23-galactosyltransferase catalysing the formation of type 1 chains. J. Biol. Chem. 273:433-440, 1998; Hennett, T., Dinter, A., Kubnert, P., Mattu, T. S., Rudd, P. M. and Berger, E. G. Genomic cloning and expression of three murine UDP-galactose: xcex2-N-acetylglucosamine/xcex21,3-galactosyltransferase genes. J. Biol. Chem. 273:58-65, 1998); (ii) although xcex23Gal-T3 has a wider expression pattern it is not detected in several tissues including colon and it is weakly expressed in gastric mucosa (Amado, M., Almeida, R., Carneiro, F., et al. A family of human xcex23-galactosyltransferases: characterisation of four members of a UDP-galactose xcex2-N-acetylglucosamine/xcex2-N-acetylgalactosamine xcex21,3-Galactosyltransferase family. J. Biol. Chem. 273:12770-12778, 1998; Kolbinger, F., Streiff, M. B. and Ktopodis, A G. Cloning of a human T.JDP-galactose:2-acetamido-2-deoxy-D-glucose xcex23-galactosyltransferase catalysing the formation of type 1 chains. J. Biol. Chem. 273:433-440, 1998); (iii) the kinetic properties of recombinant enzymes are not consistent with those reported for xcex2Gal-T activities in epithelia (Sheares, B. T., Lau, J. T. and Carlson, D. M. Biosynthesis of galactosyl-beta xcex21,3-N-acetylgiucosamine. J. Biol. Chem. 257:599-602, 1982; Holmes, E. H. Characterization and membrane organization of beta 1-3 and beta 1-4 galactosyltransferases from human colonic adenocarcinoma cell lines Cob 205 and SW403: basis for preferential synthesis of type 1 chain lacto-series carbohydrate structures. Arch Biochem Biophys 270:630-646, 1989); and (iv) the acceptor substrate specificities of xcex23Gal-T1, -T2, or -T3 do not include the mucin-type core 3 structure (Amado, M., Almeida, R., Cameiro, F., et al. A family of human xcex23-galactosyltransferases: characterisation of four members of a UDP-galactose xcex2-N-acetylglucosamine/xcex2-N-acetylgalactosamine xcex21,3-Galactosyltransferase family. J. Biol. Chem. 273:12770-12778, 1998; Hennett, T., Dinter, A., Kuhnert, P., Mattu, T. S., Rudd, P. M. and Berger, E. G. Genomic cloning and expression of three murine UDP-galactose: xcex23-N-acetylglucosamine xcex21,3-galactosyltransferase genes. J. Biol. Chem. 273:58-65, 1998), which was previously found to be a highly efficient substrate for xcex23Gal-T activity isolated from porcine trachea (Sheares, B. T. and Carison, D. M. Characterization of UDP-galactose:2-acetamido-2-deoxy-D-glucose 3 beta-galactosyltransferase from pig trachea. J. Biol. Chem. 25 8:9893-9898, 1983).
Access to additional existing xcex2GlcNAc xcex23Gal-transferase genes encoding xcex23Gal-transferases with better kinetic properties than xcex23Gal-T1, -T2, and -T3 would allow production of more efficient enzymes for use in galactosylation of oligosaccharides, glycoproteins, and glycosphingolipids. Such enzymes could be used, for example, in pharmaceutical or other commercial applications that require synthetic galactosylation of these or other substrates that are not or poorly acted upon by xcex23Gal-T1, -T2, and -T3, in order to produce appropriately glycosylated glycoconjugates having particular enzymatic, immunogenic, or other biological and/or physical properties.
Consequently, there exists a need in the art for additional isolated UDP-galactose: xcex23-N-acetyl-glucosamine xcex21-3-Galactosyltransferases having unique, specific properties and the primary structure of the genes encoding these enzymes. The present invention meets this need, and further presents other related advantages, as described in detail below.
The present invention provides isolated nucleic acids encoding human UDP-galactose: xcex23-N-acetylglucosamine xcex21,3-galactosyltransferase (xcex23Gal-T5), including cDNA and genomic DNA. xe2x8ax963Gal-T5 has better kinetic properties than xcex23Gal-T1, -T2, and T3, as exemplified by its better activity with saccharide derivatives and glycoprotein substrates as well as its activity with globoside glycolipid. Indeed, xcex23Gal-T5 is the first glycosyltransferase available for transfer of Gal xcex21-3 to globoside (GalNAcxcex21-3Galxcex11-4Galxcex21-4Glcxcex21-Cer). The complete nucleotide sequence of xcex23Gal-T5, is set forth in FIG. 1.
In one aspect, the invention encompasses isolated nucleic acids comprising or consisting of the nucleotide sequence of nucleotides 1-933 as set forth in FIG. 1, or sequence-conservative or function-conservative variants thereof. Also provided are isolated nucleic acids hybridizable with nucleic acids having the sequence as set forth in FIG. 1 or fragments thereof or sequence-conservative or function-conservative variants thereof. In various embodiments, the nucleic acids of the invention are hybridizable with xcex23Gal-T5 sequences under conditions of low stringency, intermediate stringency, high stringency, or specific preferred stringency conditions defined herein. In one embodiment, the DNA sequence encodes the amino acid sequence, as set forth in FIG. 1, from methionine (amino acid no. 1) to valine (amino acid no. 310). In another embodiment, the DNA sequence encodes an amino acid sequence comprising a sequence from methionine (no. 25) to valine (no. 310) as set forth in FIG. 1.
In a related aspect, the invention provides nucleic acid vectors comprising xcex23Gal-T5 DNA sequences, including but not limited to those vectors in which the xcex23Gal-T5 DNA sequence is operably linked to a transcriptional regulatory element (e.g. a promoter, an enhances, or both), with or without a polyadenylation sequence. Cells comprising these vectors are also provided, including without limitation transiently and stably expressing cells. Viruses, including bacteriophages, comprising xcex23Gal-T5-derived DNA sequences are also provided. The invention also encompasses methods for producing xcex23Gal-T5 polypeptides. Cell-based methods include without limitation those comprising: introducing into a host cell an isolated DNA molecule encoding xcex23Gal-T5, or a DNA construct comprising a DNA sequence encoding xcex23Gal-T5; growing the host cell under conditions suitable for xcex23Gal-T5 expression; and isolating xcex23Gal-T5 produced by the host cell. Further, this invention provides a method for generating a host cell with de novo stable expression of xcex23Gal-T5 comprising: introducing into a host cell an isolated DNA molecule encoding xcex23Gal-T5 or an enzymatically-active fragment thereof (such as, for example, a polypeptide comprising amino acids 25-310 as set forth in FIG. 1), or a DNA construct comprising a DNA sequence encoding xcex23Gal-T5 or an enzymatically active fragment thereof; selecting and growing host cells in an appropriate medium; and identifying stably transfected cells expressing xcex23Gal-T5. The stably transfected cells may be used for the production of xcex233Gal-T5 enzyme for use as a catalyst and for recombinant production of peptides or proteins with appropriate galactosylation. For example, eukaryotic cells, whether normal or diseased cells, having their glycosylation pattern modified by stable transfection as above, or components of such cells, may be used to deliver specific glycoforms of glycopeptides and glycoproteins, such as, for example, as immunogens for vaccination.
In yet another aspect, the invention provides isolated xcex23Gal-T5 polypeptides, including without limitation polypeptides having the sequence set forth in FIG. 1, polypeptides having the sequence of amino acids 25-310 as set forth in FIG. 1, and a fusion polypeptide consisting of at least amino acids 25-310 as set forth in FIG. 1 fused in frame to a second sequence, which may be any sequence that is compatible with retention of xcex23Gal-T5 enzymatic activity in the fusion polypeptide. Suitable second sequences include without limitation those comprising an affinity ligand, a reactive group, and/or a functional domain from another protein.
In another aspect of the present invention, methods are disclosed for screening for mutations in the coding region (exon I) of the xcex23Gal-T5 gene using genomic DNA isolated from, e.g., blood cells of normal and/or diseased subjects. In one embodiment, the method comprises: isolation of DNA from a normal or diseased subject; PCR amplification of coding exon I; DNA sequencing of amplified exon DNA fragments and establishing therefrom potential structural defects of the xcex23Gal-T5 gene associated with disease.
These and other aspects of the present invention will become evident upon reference to the following detailed description and drawings.